The free radical/oxidative stress field has a long history of papers devoted to lipid peroxidation and DNA oxidation, but protein oxidation and, particularly, altered proteolytic susceptibility have not been studied by very many laboratories. Reasons for this apparent reluctance to measure protein degradation as a consequence of oxidative stress may well include the difficulty, expense, and (even) danger of the available methods. Basically, until now, if one wanted to study how oxidation may change the proteolytic susceptibility of any given purified protein (or mixture of protein substrates), one needed to be willing to use radioactive labels, or tracers. For many laboratories, the complicated protein labeling techniques, radioactive isotope training and licenses or permits, radioactive waste disposal problems, potential dangers to lab workers, and high costs of radioactive techniques have proven to be major barriers to the study of protein oxidation and proteolysis.
The use of 3H and 14C labeling of proteins by in vitro reductive methylation has become an important tool to measure the proteolytic degradation of a wide range of protein substrates by purified proteolytic enzymes, cell lysates, and cell extracts. Such 3H and 14C labeled protein substrates are also widely used to assess the effects of protein modifications, such as denaturation, oxidation, methylation, acetylation, etc., on proteolytic susceptibility and rates of turnover. In addition, the specificity of various proteolytic enzymes for putative substrates has frequently been tested using 3H and 14C labeled proteins. The process of in vitro reductive methylation with 3H and 14C, however, does have a number of drawbacks. The use of radioactive materials, with all the attendant exposure risks for experimenters and their colleagues, and the difficulties and ethical considerations of radioactive waste procedures rank high on the list of drawbacks. Experimenters must maintain radioactive use permits that require frequent evidence of ongoing training and compliance. Additionally, the costs both of purchasing radionucleotides and of disposing of them are extremely high. Proteolytic assays with 3H and 14C labeled protein substrates require a labor-intensive TCA precipitation step, so that undegraded (TCA-insoluble) proteins can be separated from TCA-soluble degradation products. This further increases the volume of radioactive waste, limits the number of samples that may be analyzed, increases experimental error, and forces an absolute endpoint to the assay with the result that continuous time courses cannot be measured. These drawbacks have effectively limited the preparation and use of radio-labeled protein substrates to study protein degradation to those laboratories where proteolysis is the major topic.
Fluorometric peptidase assays, in which a fluorophore covalently linked to a small peptide sequence is cleaved by a protease/proteinase, provides a solution to all the above radiolabeling problems, and small fluorogenic peptides are widely used to measure peptidase activities. Such fluorogenic peptidase measurements are based on the increase in fluoresence as the fluorophore is released from the peptide by proteolytic cleavage. TCA precipitation is not required, thus enabling continuous readings to be made, as well as permitting a greater number of assays to be performed. While this technology has been highly valuable in measuring the cleavage of short peptide sequences, it is only a primitive model with which to test the activities of complete proteinases which target whole proteins rather than short peptide sequences. Additionally many proteinases are selective for various modified forms of their protein substrates, and such selectivity cannot be measured by peptide hydrolysis.
A solution would seem to be that of adapting the fluorescence labeling technique for peptides to work with intact proteins, but there has been limited success in modifying this technology to measure the degradation of whole proteins. Two techniques have been described for attaching fluorophores onto proteins. FITC labeling has been used to label casein, hemoglobin (Hb), and bovine serum albumin (BSA). However, FITC-labeled proteins are highly unstable and so must be precipitated and stored in 50% ammonium sulfate and then transferred out of solution, just before use. These steps are major drawbacks and present considerable contamination risks as well as limiting the time over which assays can be performed. The assay is further limited by a strong dependency on pH for the sensitivity of the fluorophore, making assays of strongly acidic proteases such as pepsin, or strongly alkaline proteases such as proteinase K, impractical. In addition, for measuring proteolysis, this technique is, like radiolabeling, limited by the requirement for TCA precipitation, which makes it labor intensive, error prone, and extremely limited to small-size experiments. The second technique involves labeling of either casein or BSA with BODIPY. This technique provides a number of advantages over both FITC labeling and radiolabeling, though it also has several drawbacks. For example, BODIPY has a very small separation between excitation and emission wavelengths (503 nm/512 nm) compared to other fluorophores such as 7-amino-4-methylcoumarin (AMC; 365 nm/444 nm), which makes it extremely difficult to detect the signal without highly specialized equipment. The label is relatively large and complex (389-634 Da, depending on type of BODIPY label) compared to the small [3H]formaldehyde label (32 Da) used in radiolabeling; this raises some concerns about modification of the protein during BODIPY labeling. BODIPY is also relatively expensive for very small quantities, compared with other fluorophores. Finally, there are only a small number of assays for which BODIPY has been described. Thus, most studies of protein degradation continue to rely on in vitro radiolabeling ([3H] or [14C]) of purified protein substrates, using the technique of reductive methylation developed by Jentoft and Dearborn (Jentoft, N.; Dearborn, D. G. Labeling of proteins by reductive methylation using sodium cyanoborohydride. J. Biol. Chem. 254:4359-4365; 1979).
Accordingly, since in vitro radio-labeling of protein substrates is desirably avoided and neither FITC—nor the BODIPY-labeling alternatives appear entirely suitable, there is a need for improved methods of labeling protein substrates.